Integrins, extracellular matrix molecules, and cytoskeletal proteins contribute to cell adhesion and migration, and they participate in cell surface control of tissue organization, gene expression, and growth. [unreadable] We are addressing the following general questions:[unreadable] 1. How are integrins, the extracellular matrix, and the cytoskeleton integrated, and how is their regulatory crosstalk coordinated to produce cell migration?[unreadable] 2. What are the subcellular structures and signaling pathways involved in cell migration?[unreadable] [unreadable] We are using a variety of cell and molecular biology approaches to address these questions, including biochemical analyses, fluorescent chimeras, fluorescence time-lapse microscopy, and mutational analysis. We have generated a variety of fluorescent molecular chimeras and mutants of cytoskeletal proteins, including paxillin, vinculin, talin, tensin, and zyxin, as part of a long-term program to analyze their functions in integrin-mediated processes. We have been focusing particularly on functions of integrins and associated extracellular and intracellular molecules in the mechanisms and spatial regulation of cell migration. [unreadable] [unreadable] Nonmuscle cellular myosins and actin are thought to play crucial roles in cell migration and in many developmental and wound repair processes, but the roles of the major myosin IIA gene were not clear. We recently published a study investigating the roles of the major myosin II genes, myosin IIA and IIB. One set of findings was not unexpected: we found that myosin IIA plays central roles in fibroblast and embryonic stem cell contractility, actin cytoskeletal organization, and organization of cell-matrix adhesions. However, unexpectedly and in contradiction to the belief that myosin II molecules were needed for cell migration, we showed that myosin IIA is not required, and in fact that it serves as a brake on migration. Particularly surprisingly, however, we discovered strong cross-regulation between myosin IIA and microtubule dynamics that regulates Rac localization and cell migration. One puzzling finding was our evidence implicating kinesin 5, a mitotic kinesin, in this crosstalk, which needs further examination. For technical reasons, it was necessary in our original study to use standard two-dimensional culture systems for visualizing microtubule dynamics. A new project will examine how similar these principles are in three-dimensional systems, since other properties of cells can differ. [unreadable] [unreadable] A particularly intensively studied mediator of integrin cytoskeletal and signaling interactions is the scaffold protein termed focal adhesion kinase (FAK). We had previously published a series of studies on FAK concerning its potential roles in adhesion and migration in normal and tumor cells. We are continuing a collaboration with Hilary Beggs at UCSF and Joseph Schlessinger at Yale to determine roles of FAK and its homologue Pyk2 in cell migration in two- and three-dimensional microenvironments and other processes using conditional knockout cells. [unreadable] [unreadable] We and others have been exploring the mechanisms guiding the directionality of cell migration. Besides externally directed migration, we had previously described an intracellular signaling mechanism based on total level of the small GTPase Rac1. Cells migrating in a three-dimensional matrix displayed a moderate decrease in total Rac activity associated with directionally persistent migration, and this behavior could be mimicked in two-dimensional cell culture by reducing Rac levels or activity. However, an addition mechanism guiding directional cell migration is known to be interaction with cues from the migratory substrate, e.g. migration along extracellular matrix fibrils. We are examining this phenomenon in depth to test whether aspects of three-dimensional matrix guidance of migration can be mimicked by a one-dimensional linear matrix pattern on a substrate in regulating the nature of cell adhesions, cytoskeletal organization and function, and the mode and efficiency of cell migration.[unreadable] [unreadable] These ongoing studies on the functions of integrins and associated intracellular and extracellular molecules in cell migration center upon our ability to image live-cell molecular dynamics of early cell protrusions and intracellular myosin and microtubules. All of these processes will need to be analyzed in parallel in real time and in physiological matrix environments to be able to understand the mechanisms of in vivo cell migration. This combined knowledge should provide novel approaches to understanding, preventing, or ameliorating migratory processes that cells use in abnormal development and cancer. An in-depth understanding of exactly how cells move and interact with their matrix environment will also facilitate tissue engineering studies.